In most cell types, HIV-1 assembly occurs predominantly on the plasma membrane; however, the itinerary that the Gag precursor follows to reach its destination in the cell remains ill-defined. It has been suggested that even in cell types in which assembly and budding occur predominantly at the plasma membrane, Gag traffics through an endosomal or endocytic compartment before reaching the cell surface. We and others have demonstrated that the matrix (MA) domain of Gag regulates the site of virus assembly, and we discovered recently that a member of the phosphoinositide family of lipids, phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2], serves an important function in directing Pr55Gag to the plasma membrane. We are actively engaged in studies that seek to define the subcellular site of HIV-1 assembly in physiologically relevant cell types, and to elucidate further the cellular machinery involved in HIV-1 Gag trafficking. This effort makes use of live-cell imaging techniques recently developed in the VCIS, and is also being extended to the non-human primate lentiviruses equine infectious anemia virus (EIAV) and feline immunodeficiency virus (FIV). The viral envelope (Env) glycoproteins are incorporated into virions during the assembly process. We have shown that point mutations in the MA domain of Gag block Env incorporation, and demonstrated that the long gp41 cytoplasmic tail plays a cell type-dependent role in Env incorporation. Despite the evidence for a direct interaction between MA and the gp41 cytoplasmic tail, the molecular mechanism by which Env is incorporated, the subcellular site of initial GagEnv contact, and the basis for cell type dependence of cytoplasmic tail function remain to be defined. We will address these issues; specifically, we will evaluate the role of cellular factors and lipid rafts in Env incorporation. [Corresponds to Freed Project 1 in the April 2007 site visit report of the HIV Drug Resistance Program]